Hoechst 342

The plasma membrane integrity and mitochondrial activity in fresh and frozen–thawed samples were examined simultaneously through the combined use of fluorophores Hoechst 342 (H342; Sigma-Aldrich, St. Louis, MO, USA), Mito Tracker red^® (CMXRos, Molecular Probes, M-7512) and propidium iodide (IP, Sigma-Aldrich, Co., St. Louis, MO, USA). A total of 200 cells were counted under epifluorescence microscopy (EFA fluorescent accessory Halogen Lamp Set. Leica. Kista, Sweden). Cells that had a head marked in blue (H-342) were classified as having an intact membrane, while those that had a head marked in red (PI) were identified as having a nonintact membrane. In addition, those with the intermediate piece marked in red were considered to have an active mitochondrial function [17 (link),19 (link)].

Acinar cells were isolated and seeded into CellCarrier Ultra 96-well microplates (Perkin Elmer, Waltham, MA, USA). Cells were treated with TCT and cerulein or hydrogen peroxide for 24 h. Cells were then stained with propidium iodide (2.5 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA) and Hoechst342 (5 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Images were analyzed as previously reported [52 (link)] with an Opera Phenix High-Content Analysis system (Perkin Elmer, Waltham, MA, USA) using a 40× air objective and appropriate laser and filter settings in sequential mode to avoid overlapping of the emission spectra.